In vivo CRISPR screen identifies tumor suppressors ABSTRACT# as drivers of tumor cell-intrinsic immune evasion 1905 Chengyin Min1, Ferdinandos Skoulidis2, Wenrong Zhou3, Jennifer Tsoi1, Xuewen Pan1, Alan Huang1, Matthew Goldstein1 1Tango Therapeutics, Cambridge, USA. 2The University of Texas MD Anderson, Houston, USA. 3WuXi AppTec Group, Shanghai, China.

Tumor growth reflects increased anti-tumor STK11 loss drives a cold and suppressive ABSTRACT STK11 knockout drives immune evasion immunity in vivo Stk11 vs NTC myeloidimmune environment A B 100 Cancer cell-intrinsic genetic alterations that allow cancer cells to evade G7 sgNTC anti-IgG2a destruction by the immune system remain poorly characterized. Here we performed a pooled CRISPR-Cas9-based in vivo genetic screen targeted to a 80 G8 sgNTC anti-PD1

pre-defined set of tumor suppressor to mimic loss-of-function +

5 G6 sgStk11 anti-IgG2a in syngeneic tumor models. CRISPR edited tumor cells were implanted into 4 60

immune-deficient or immune-competent C57BL/6 mice, a subset of which were D G1 sgStk11 anti-PD1

C

treated with anti-PD1 to simulate increased immune pressure. Tumor samples f

o 40 at the endpoint were subjected to next generation sequencing analysis to identify tumor suppressor genes driving immune evasion. The screen % confirmed previously identified immunotherapy targets such as CD47 and Adar 20 as well as known drivers of immune resistance in the interferon signaling and antigen presentation pathways. Importantly, we identified loss of STK11 and 0 KEAP1 as drivers of immune resistance in syngeneic models. TIL profiling id C C C e o D S S g analysis suggests that STK11 knockout induces a “cold” tumor l + D D a e b h microenvironment. STK11 and/or KEAP1 genomic alterations are found in y 1 -M -M p M 1 ro D N M c ~25% of non-squamous non-small cell and have been reported as C M a P M a major predictor of primary resistance to PD-1 blockade. Together, these data 3LL3-sLgLN-sTgCN-aTlCl--inal-lo-inne-one demonstrate that high-throughput in vivo genetic screens can identify tumor 4000 Figure 3: Tumors grow slower with increasing immune pressure in vivo. Mice were grouped ) 4000

cell-intrinsic drivers of immune evasion and establish murine system relevant ) 3

to the study of primary resistance to PD-1 axis immunotherapy and more on day 0 when average tumor volumes reach 50mm3. Average tumor volumes for each condition 3 m

generally, tumor cell-intrinsic immune evasion. were plotted. Gradually decreasing tumor growth from nude mice to C57BL/6 treated with anti-IgG m

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to C57BL/6 mice treated with anti-PD1 reflects increased anti-tumor immunity in these three in vivo ( 3000

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conditions. e

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CRISPR tumor suppressor library u

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Positive controls work well in the screen o

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Additional genes Essential genes 100o 01000

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u T Immune positive controls T 0 0 0 0 5 5 10 10 15 15 Figure 6: STK11 knockout drives resistance to immune pressure in vivo. 3LL cells were transduced with sgRNAs targeting STK11D ora nonysD- targetingpaoyss tp to rcontrolesat ttmr e(NTC).eantmt ien Derivativesnitti aintiiotina weretion inoculated into C57BL6 mice treated with anti-PD1 or anti-IgG2a or anti-CD4/CD8 to deplete T cells. Depletion of STK11 drives resistance to increasing immune pressure in vivo. G1: 3GL1L: p3aLrLe nptaarle anntatil- CanDt4i-/CCDD48/CD8 Stk11 vs NTC ratio Negative controls G2: 3GL2L: p3aLrLe nptaarle anntatil- IagnGti2-IagG2a 15 KEAP1 knockout drives immune evasion G7 sgNTC anti-IgG2a G3: 3GL3L: p3aLrLe nptaarle anntatil- PanDt1i-PD1 G8 sgNTC Genes with loss-of- Pan-cancer G4: sGg4K:E sAgPK1E AaPnt1i- CanDt4i-/CCDD48/CD8 anti-PD1 deleted genes 10 function mutations G5: sgKEAP1 anti-IgG2a o

G5: sgKEAP1 anti-IgG2a i G6 sgStk11 anti-IgG2a

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G6: sGg6K:E sAgPK1E AaPnt1i- PanDt1i-PD1 R G1 n = 162 genes; 10sgRNAs/ G7: sGg7N: TsCgN aTnCti- CanDt4i-/CCDD48/CD8 5 sgStk11 Figure 4: Positive control genes were depleted as expected. sgRNAs for , an essential anti-PD1 gene, were depleted in all conditions relative to plasmid pool. sgRNAs targeting Cd47, a well- G8: sGg8N:T sCg NaTnCti- IagnGti2-IagG2a known immune target for macrophages, were depleted as expected in all in vivo conditions. G9: sgNTC anti-PD1 In vivo screen design G9: sgNTC anti-PD1 0 STK11 and KEAP1 are among the top resistant CD8T/Treg CD4Teff/Treg

genes to immune pressure Figure 8: STK11 knockout drives a suppressive immune environment in vivo. Immunophenotyping analysis were conducted on sgNTC vs sgStk11 tumors upon anti-PD1 or anti-IgG2a treatment. Stk11 KO increased the frequency of MDSCs, decreased CD3 T cells and CD8 T cells and CD8TEM (effector memory T cells) in the tumor immune environment. Nude mice SUMMARY

• A pooled CRISPR-Cas9-based in vivo screen was performed C57BL/6 mice successfully in conditions reflecting increased anti-tumor immunity 3LL cells infected • STK11 and KEAP1 were identified as immune evasion contexts with sgRNA library C57BL/6 mice • Depletion of STK11 or KEAP1 drives resistant to immune pressure + anti-PD1 in immune competent mice

• Immunophenotyping analysis comparing STK11 knockout tumors vs control indicated STK11 loss drives a cold and suppressive Figure 2: Diagram of in vivo screen platform and design. 3LL cells were transduced with Figure 5: STK11 and KEAP1 are among the top resistant genes from the screen. sgRNA Figure 7: KEAP1 knockout drives resistance to immune pressure in vivo. 3LL cells were immune environment CRISPR tumor suppressor library and stable cell line generated by puromycin selection. Stable depletion or enrichment from tumors of C57BL/6 mice + anti-PD1 vs. C57BL/6 mice + anti-IgG is transduced with sgRNAs targeting KEAP1 or non-targeting control (NTC). Derivatives were inoculated cells were inoculated into nude mice, C57BL/6 mice and C57BL/6 mice treated with anti-PD1 represented in a volcano plot. sgRNAs for Stk11 and Keap1 are the top two most enriched in C57BL/6 into C57BL6 mice treated with anti-PD1 or anti-IgG2a or anti-CD4/CD8 to deplete T cells. Depletion of representing increasing degrees of anti-tumor immunity. treated with anti-PD1 vs anti-IgG whereas sgRNAs for Ptpn2 were among the most depleted. KEAP1 drives resistance to increasing immune pressure in vivo.